JP2019042280A - Elastic fibrous tissue regeneration substrate and method for producing same - Google Patents

Abstract

To provide an elastic fibrous tissue regeneration substrate that can promote regeneration of elastic fibrous tissue by transplantation into a living body, and to provide a method for producing said elastic fibrous tissue regeneration substrate.SOLUTION: Provided is an elastic fibrous tissue regeneration substrate comprising LTBP-4 protein and cross-linked collagen sponge carrying said LTBP-4 protein, and a method for producing elastic fibrous tissue regeneration substrate in which a collagen solution containing LTBP-4 protein is impregnated into cross-linked collagen sponge and then freeze-dried.SELECTED DRAWING: None

Description

The present invention relates to an elastic fibrous tissue regeneration substrate capable of promoting regeneration of elastic fibrous tissue by transplantation into a living body, and a method of manufacturing the elastic fibrous tissue regeneration substrate.

Among human body tissues, tissues such as lungs, blood vessels, and skin are highly elastic and stretchable tissues, but the elasticity of body tissues such as hardening of blood vessels with age or sagging of skin is lost with aging. An aging phenomenon occurs. It is considered that this is because the elastic fiber present in the living tissue, which is a fibrous tissue having flexibility and elasticity like rubber, is deteriorated. It is believed that this elastic fiber is not regenerated even if it is degraded or degraded, and many studies have been made on the regeneration of such an elastic fiber.

Elastic fibers are formed by depositing elastin protein in microfibrils (Fibrillin-1, 2 homopolymer) and crosslinking with lysyl oxidase enzyme. A protein known to co-localize in this microfibril is the LTBP (latent TGFβ-binding protein) family. To date, the presence of four family members of LTBP 1-4 protein has been identified in this family. Patent Document 1 discloses an elastic fiber regenerating agent containing LTBP-4 protein.

JP, 2011-026236, A

In Patent Document 1, the formation of an elastic fiber tissue was confirmed by culturing human fibroblasts seeded in a 24-well plate using an elastic fiber regenerating agent containing LTBP-4 protein. However, since an actual living tissue has a three-dimensional structure, it is important to regenerate an elastic fibrous tissue having a three-dimensional structure. In addition, it has also been an issue as to what form an elastic fiber regenerating agent containing LTBP-4 protein can be applied to a living body to promote the regeneration of elastic fiber tissue.
The present invention provides an elastic fiber tissue regeneration base material capable of promoting regeneration of elastic fiber tissue by implanting in a living body in view of the above-mentioned present situation, and a method for producing the elastic fiber tissue regeneration base material. To aim.

The present invention is an elastic fibrous tissue regeneration substrate comprising LTBP-4 protein and a crosslinked collagen sponge carrying the LTBP-4 protein.
The present invention will be described in detail below.

As a result of intensive studies, the inventor of the present invention has found that the elastic fiber tissue regeneration base material on which LTBP-4 protein is supported on a crosslinked collagen sponge can infiltrate and proliferate fibroblasts and the like when it is transplanted into a living body. It has been found that the formation of elastic fiber tissue can be promoted by the -4 protein, and the present invention has been completed.

The elastic fiber tissue regeneration substrate of the present invention comprises LTBP-4 protein and a crosslinked collagen sponge carrying the LTBP-4 protein. By supporting the crosslinked collagen sponge, the LTBP-4 protein can be stably held and transplanted to a living body, and the formation of elastic fibrous tissue can be promoted. In addition, since the implanted crosslinked collagen sponge gradually degrades, LTBP-4 protein can be slowly released according to the degradation.

The cross-linked collagen sponge is a substrate for carrying LTBP-4 protein, and a scaffold for fibroblasts and the like to invade and proliferate, and promote elastic fiber tissue by LTBP-4 protein. Have a role.

It does not specifically limit as collagen used as a raw material of the said crosslinked collagen sponge, For example, what originates in skins, such as cows and pigs, etc., a tendon, etc. can be used. From the viewpoint of removing antigenicity and enhancing safety, it is preferable to use atelocollagen in which collagen is treated with an enzyme such as a protease or pepsin to remove telopeptides as much as possible. There are I-IV types for atelocollagen, which can be appropriately selected.

In the crosslinked collagen sponge, the lower limit of the average pore diameter is preferably 0.1 μm, and the upper limit is preferably 500 μm. When the average pore size is less than 0.1 μm, fibroblasts and the like can not penetrate into the elastic fiber tissue regeneration substrate, and it becomes difficult to regenerate sufficient elastic fiber tissue. When the average pore size exceeds 500 μm, the density of invading fibroblasts and the like becomes insufficient, and elastic fiber tissue becomes difficult to be regenerated. A more preferable lower limit of the average pore diameter is 1 μm, a more preferable upper limit is 300 μm, a still more preferable lower limit is 5 μm, and a still more preferable upper limit is 100 μm.

The thickness of the crosslinked collagen sponge is not particularly limited, but a preferable lower limit is 0.1 mm and a preferable upper limit is 3 mm. If the thickness is less than 0.1 mm, an elastic fiber tissue having a sufficient thickness may not be formed or the handleability at the time of transplantation may be inferior, and if it exceeds 3 mm, the invaded fibroblasts It may be inferior to nutrient supply to cells etc.

The method for producing the crosslinked collagen sponge is not particularly limited. For example, the non-crosslinked collagen sponge obtained by a method such as vacuum freeze-drying of an aqueous collagen solution is subjected to heat crosslinking, UV crosslinking or chemical crosslinking using a crosslinking agent. And the like.
Specifically, for example, collagen adjusted to preferably 0.1 to 1% by weight, more preferably 0.1 to 0.5% by weight with distilled water, physiological saline, phosphate buffered saline (PBS), etc. The solution is lyophilized to produce an uncrosslinked collagen sponge. At this time, a very small amount of a lipid-soluble organic solvent such as chloroform may be added to the collagen solution, or the collagen solution may be homogenized and foamed. Conditions for lyophilization are not particularly limited. For example, the collagen solution is cooled to -135 to -40 ° C and frozen, and lyophilization is performed for 6 to 24 hours while raising the temperature from -40 to 40 ° C. it can. A cross-linked collagen sponge is obtained by subjecting the obtained non-cross-linked collagen sponge to a cross-linking treatment such as thermal cross-linking, ultraviolet cross-linking, chemical cross-linking and the like. The crosslinked collagen sponge can exhibit a function as a scaffold for forming an elastic fibrous tissue because its biodegradation rate is adjusted. The thermal crosslinking can be carried out, for example, by treating a collagen sponge at about 100 to 150 ° C. for about 6 to 24 hours using a vacuum dryer. The chemical crosslinking can be performed, for example, by immersing a collagen sponge in a glutaraldehyde solution. Specifically, by immersing in a 0.1 to 1% (especially 0.2%) glutaraldehyde solution for 1 to 50 hours (especially 3 to 15 hours) at 0 to 10 ° C (especially around 4 ° C) It can be implemented.

The LTBP-4 protein is a member of the LTBP (latent TGFβ-binding protein) family, which is known as a protein colocalized in microfibrils.
In the present invention, the LTBP-4 protein is derived from the base sequence (GenBank accession number AF051344) described in SEQ ID NO: 1 disclosed in Patent Document 1 or an orthologue thereof, or a variant thereof (SNP, including haplotype) Polypeptide refers to the The orthologue of LTBP-4 protein is not particularly limited, and may be derived from, for example, any animal, preferably a mammal. Examples of mammals include cows, sheep, pigs, goats, monkeys, rabbits, rats, hamsters, guinea pigs, mice and the like. The LTBP-4 protein is a secreted protein and processing may remove the signal sequence (eg, MAGGVRLLWVSLLVLLAQLGPQPGLG). In the present invention, any LTBP-4 protein from which a signal sequence has been removed or from which a signal sequence has not been removed can be used, but those from which a signal sequence has been removed are preferred. In addition, the LTBP-4 mutant is not particularly limited as long as it enables elastic fiber regeneration, and has a sequence complementary to the base sequence shown in SEQ ID NO: 1 disclosed in Patent Document 1, for example Examples include polypeptides derived from DNA that hybridizes with DNA under stringent conditions and encodes LTBP-4.
As for LTBP-4 protein, in addition to Patent Document 1, for example, “Tomo Nakajima, LTBP-4 essential for elastic fiber formation, Biochemistry, vol. 86, No. 6, pp. 770-773 (2014)”, etc. It is described in detail.

The LTBP-4 protein is carried by the crosslinked collagen sponge. Thereby, the LTBP-4 protein can be stably retained to promote formation of elastic fiber tissue when the elastic fiber tissue regeneration substrate of the present invention is implanted. In addition, since the implanted crosslinked collagen sponge gradually degrades, LTBP-4 protein can be slowly released according to the degradation.

The content of the above LTBP-4 protein in the elastic fiber tissue regeneration substrate of the present invention is not particularly limited, but the preferable lower limit is 0.08% by weight, and the preferable upper limit is 8.0% by weight. If the content of LTBP-4 protein is within this range, formation of elastic fiber tissue can be surely promoted. The more preferable lower limit of the content of the LTBP-4 protein is 0.16% by weight, and the more preferable upper limit is 1.6% by weight.

The elastic fiber tissue regeneration substrate of the present invention preferably further comprises a DANCE (Developmental Architecture and Neural Crest Epidermal growth factor (EGF) -like; also referred to as fibriln-5) protein. By combining DANCE protein with LTBP-4 protein, formation of elastic fiber tissue can be further promoted.
Here, the "DANCE protein" is a secreted protein also called fibriln-5, and is known to be a molecule that constitutes an elastic fiber. DANCE proteins include human DANCE proteins of human origin, or orthologues thereof, or variants thereof (including SNPs and haplotypes). The orthologue of the DANCE protein is not particularly limited, and may be derived from, for example, any animal, preferably a mammal. Examples of mammals include cattle, sheep, pigs, goats, monkeys, rabbits, rats, hamsters, guinea pigs, mice and the like.
The DANCE protein is described in detail, for example, in WO 2006/082763.

The content of the DANCE protein in the elastic fiber tissue regeneration substrate of the present invention is not particularly limited, but the preferred lower limit is 0.001% by weight, and the preferred upper limit is 1% by weight. When the content of DANCE protein is in this range, formation of elastic fiber tissue can be further promoted when transplanted. A more preferable lower limit of the content of the DANCE protein is 0.01% by weight, and a more preferable upper limit is 0.8% by weight.

The elastic fibrous tissue regeneration substrate of the present invention may further contain mucopolysaccharides. By including the mucopolysaccharide, it is possible to control the sustained release rate of LTBP-4 protein and DANCE protein from the substrate. This is considered to be because, since the mucopolysaccharide has a hydrophilic group, the moisture content can be controlled by controlling the amount of the hydrophilic group of the substrate by the addition amount of the mucopolysaccharide.
Examples of the mucopolysaccharides include chondroitin sulfate, heparin, hyaluronic acid, dermatan sulfate, keratan sulfate, heparan sulfate and the like. Among them, chondroitin sulfate and heparin are preferable in terms of solubility with collagen and availability at low cost.

The content of the mucopolysaccharide in the elastic fiber tissue regeneration substrate of the present invention is not particularly limited, but a preferable lower limit is 0.1% by weight, and a preferable upper limit is 50% by weight. When the content of mucopolysaccharide is within this range, the sustained release rate of LTBP-4 protein and the like can be controlled with certainty. A more preferable lower limit of the content of the mucopolysaccharide is 1% by weight, and a more preferable upper limit is 20% by weight.

Although the method for producing the elastic fiber tissue regeneration substrate of the present invention is not particularly limited, a collagen solution containing the above LTBP-4 protein (the above-mentioned DANCE protein and mucopolysaccharide as needed) in the above-mentioned crosslinked collagen sponge It is preferable to impregnate and then lyophilize. According to such a production method, the LTBP-4 protein and the like can be reliably supported on the crosslinked collagen sponge. Moreover, since it is not necessary to perform the process which bridge | crosslinks a collagen sponge after making the said LTBP-4 protein etc. carry | support, there is no possibility that LTBP-4 protein etc. may deactivate by crosslinking process. Therefore, sufficient strength as a scaffold for tissue regeneration, biodegradable rate, and stable loading of LTBP-4 protein can be compatible.
A method for producing an elastic fiber tissue regeneration substrate, in which a crosslinked collagen sponge is impregnated with a collagen solution containing LTBP-4 protein and then freeze-dried is also one of the present invention.

Specifically, in the method for producing an elastic fiber tissue regeneration substrate of the present invention, first, a collagen solution containing LTBP-4 protein or the like (hereinafter, also referred to as “protein-containing collagen solution”) is added to the crosslinked collagen sponge. .) Is impregnated. The specific method for impregnating is not particularly limited. For example, a method (for example, a ring) is provided at a specific place on the crosslinked collagen sponge to receive the solution, and the protein-containing collagen solution is added thereto. Can be mentioned. Alternatively, the crosslinked collagen sponge may be immersed in the protein-containing collagen solution.

It does not specifically limit as collagen used for the said protein containing collagen solution, The thing similar to the collagen used as the raw material of the said crosslinked collagen sponge can be used.
The concentration of collagen in the protein-containing collagen solution is not particularly limited, and is preferably about 0.1 to 1% by weight, and more preferably about 0.1 to 0.5% by weight.
The solvent used for the protein-containing collagen solution is not particularly limited, and examples thereof include water (distilled water), phosphate buffered saline (PBS), Carmody buffer, Tris buffer and the like. Among these, water (distilled water) is preferable because impurities are not mixed.

When the elastic fiber tissue regeneration substrate of the present invention contains mucopolysaccharides, mucopolysaccharides may be added directly to the collagen solution, but from the viewpoint of handling, the mucopolysaccharides are distilled water, physiological saline It is preferable to add a solution dissolved in a solvent such as water or phosphate buffered saline (PBS) to a collagen solution.

Next, in the method for producing an elastic fiber tissue regeneration substrate of the present invention, the crosslinked collagen sponge impregnated with the above-mentioned protein-containing collagen solution is lyophilized.
Conditions for lyophilization are not particularly limited. For example, the collagen solution is cooled to -135 to -40 ° C and frozen, and lyophilization is performed for 6 to 24 hours while raising the temperature from -40 to 40 ° C. it can.

The elastic fibrous tissue regeneration substrate of the present invention can promote the regeneration of elastic fibrous tissue by implanting in a living body.
The elastic fibrous tissue regeneration substrate of the present invention may be directly implanted at a site where elastic fibrous tissue of a living body is to be regenerated. In this case, fibroblasts and the like intrude and proliferate in the implanted elastic fiber tissue regeneration substrate to form elastic fiber tissue.
An elastic fiber tissue can also be formed by seeding and culturing fibroblasts on the elastic fiber tissue regeneration substrate of the present invention. By implanting the elastic fibrous tissue regeneration base on which the elastic fibrous tissue is formed in a site where the elastic fibrous tissue of the living body is to be regenerated, it is expected that the elastic fibrous tissue can be regenerated earlier.

According to the present invention, it is possible to provide an elastic fibrous tissue regeneration substrate capable of promoting regeneration of elastic fibrous tissue by implanting in a living body, and a method of manufacturing the elastic fibrous tissue regeneration substrate.

It is an Elastin immunostaining image of the frozen section obtained by seeding and culturing a fibroblast to the elastic fiber tissue regeneration base material obtained in Example 1 and Comparative Example 1.

EXAMPLES The embodiments of the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

Example 1
A collagen solution (0.2 wt%, pH 3) was prepared using collagen (Cellmatrix Type I-P, manufactured by Nitta Gelatin Co., Ltd.) as a raw material. The 0.2% collagen solution is frozen at -135 ° C, and then the temperature is raised from -40 ° C to 40 ° C in a vacuum dryer (0.1 MPa) with a freeze dryer to lyophilize the collagen sponge. Obtained.
The resulting collagen sponge was thermally crosslinked by heating at 140 ° C. for 14 hours to obtain a crosslinked collagen sponge.
The average pore size of the obtained crosslinked collagen sponge was about 15 μm.

An 8 mm diameter polypropylene ring was adhered to the resulting crosslinked collagen sponge. In a polypropylene ring, 80 μL of a 0.2 wt% collagen solution containing LTBP-4 protein to a concentration of 32 μg / mL was added. This is frozen at -135 ° C, and then heated from -40 ° C to 40 ° C under a reduced pressure (0.1 MPa) using a lyophilizer and freeze-dried for 14 hours to obtain an elastic fiber with a thickness of 1 mm. A tissue regeneration substrate was obtained.

(Example 2)
A collagen solution (0.2 wt%, pH 3) was prepared using collagen (Cellmatrix Type I-P, manufactured by Nitta Gelatin Co., Ltd.) as a raw material. The 0.2% collagen solution is frozen at -135 ° C, and then the temperature is raised from -40 ° C to 40 ° C in a vacuum dryer (0.1 MPa) with a freeze dryer to lyophilize the collagen sponge. Obtained.
The resulting collagen sponge was thermally crosslinked by heating at 140 ° C. for 14 hours to obtain a crosslinked collagen sponge.
The average pore size of the obtained crosslinked collagen sponge was about 15 μm.

An 8 mm diameter polypropylene ring was adhered to the resulting crosslinked collagen sponge. In a polypropylene ring, 80 μL of a 0.2 wt% collagen solution containing LTBP-4 protein and DANCE protein was added to a concentration of 32 μg / mL and 32 μg / mL, respectively. This is frozen at -135 ° C, and then heated from -40 ° C to 40 ° C under a reduced pressure (0.1 MPa) using a lyophilizer and freeze-dried for 14 hours to obtain an elastic fiber with a thickness of 1 mm. A tissue regeneration substrate was obtained.

(Comparative example 1)
A collagen solution (0.2 wt%, pH 3) was prepared using collagen (Cellmatrix Type I-P, manufactured by Nitta Gelatin Co., Ltd.) as a raw material. After freezing this 0.3% collagen solution at -135 ° C, collagen sponge is raised by raising the temperature from -40 ° C to 40 ° C under a reduced pressure (0.1 MPa) with a lyophilizer and freeze-drying it. Obtained.
The resulting collagen sponge was thermally crosslinked by heating at 140 ° C. for 14 hours to obtain a crosslinked collagen sponge.
The average pore size of the obtained crosslinked collagen sponge was about 15 μm.

An 8 mm diameter polypropylene ring was adhered to the resulting crosslinked collagen sponge. In a polypropylene ring, 80 μL of a 0.2 wt% collagen solution was added. This is frozen at -135 ° C, and then heated from -40 ° C to 40 ° C under a reduced pressure (0.1 MPa) using a lyophilizer and freeze-dried for 14 hours to obtain an elastic fiber with a thickness of 1 mm. A tissue regeneration substrate was obtained.

(Evaluation)
The formation of an elastic fiber tissue using the elastic fiber tissue regeneration substrate obtained in Example 1 and Comparative Example 1 was evaluated by the following method.
Human fibroblasts obtained by treating human skin were cultured in a 10% calf serum-added DMEM medium with fibroblasts, and cells that became confluent were harvested by trypsin treatment. The obtained human fibroblasts were suspended in DMEM / F12 medium supplemented with 10% bovine serum to prepare a fibroblast suspension.
The fibroblast suspension becomes 1 × 10 ⁶ cells inside the ring made of polypropylene of diameter 8 mm of the elastic fiber tissue regeneration substrate obtained in Example 1 and Comparative Example 1 Sowed.
Thereafter, the cells were cultured at 37 ° C. for 14 days in a CO ₂ incubator.

After culture, the elastic fiber tissue regeneration substrate was fresh, unfixed and frozen, frozen sections were prepared, and after acetone fixation, immunostaining of the elastic fiber component Elastin was performed. The Elastin immunostaining image at the time of using the elastic fiber tissue regeneration base material obtained in Example 1 is shown in FIG. 1 (a), and the Elastin immunity at the time of using the elastic fiber tissue regeneration base material obtained in Comparative Example 1 The stained image is shown in FIG. 1 (b).
From FIG. 1, when using the elastic fiber tissue regeneration base material obtained in Example 1 containing LTBP-4 protein, using the elastic fiber tissue regeneration base material obtained in Comparative Example 1 not containing It can be seen that the Elastin staining area is larger than that of.

According to the present invention, it is possible to provide an elastic fibrous tissue regeneration substrate capable of promoting regeneration of elastic fibrous tissue by implanting in a living body, and a method of manufacturing the elastic fibrous tissue regeneration substrate.

Claims (4)

An elastic fibrous tissue regeneration substrate comprising LTBP-4 protein and a crosslinked collagen sponge carrying the LTBP-4 protein. The elastic fiber tissue regeneration substrate according to claim 1, characterized in that it comprises DANCE protein. The elastic fiber tissue regeneration base according to claim 1 or 2, which comprises mucopolysaccharides. A method for producing an elastic fibrous tissue regeneration substrate, which comprises impregnating a crosslinked collagen sponge with a collagen solution containing LTBP-4 protein and then freeze-drying it.